A Method of Permeabilization of &lt;em&gt;Drosophila&lt;/em&gt; Embryos for Assays of Small Molecule Activity

Rand, Matthew D.

doi:10.3791/51634

Cited by 11 publications

(9 citation statements)

References 14 publications

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“…For embryos, the multilayered eggshell provides a formidable barrier to exogenously applied reagents: although the outer chorion can be physically or chemically removed, the waxy layer surrounding the inner vitelline membrane is essentially impermeable to most molecules. Some techniques for permeabilization exist [114, 115], and recently have been greatly improved to allow application of small molecules to embryos [116, 117]; however, they are not yet routinely used.…”

Section: Studying Embryonic Lipid Dropletsmentioning

confidence: 99%

As the fat flies: The dynamic lipid droplets of Drosophila embryos

Welte

2015

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

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Research into lipid droplets is rapidly expanding, and new cellular and organismal roles for these lipid storage organelles are continually being discovered. The early Drosophila embryo is particularly well suited for addressing certain questions in lipid-droplet biology and combines technical advantages with unique biological phenomena. This review summarizes key features of this experimental system and the techniques available to study it, in order to make it accessible to researchers outside this field. It then describes the two topics most heavily studied in this system, lipid-droplet motility and protein sequestration on droplets, discusses what is known about the molecular players involved, points to open questions, and compares the results from Drosophila embryo studies to what it is known about lipid droplets in other systems.

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Section: Studying Embryonic Lipid Dropletsmentioning

confidence: 99%

As the fat flies: The dynamic lipid droplets of Drosophila embryos

Welte

2015

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

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“…4 hours-Stage 8-9), and incubated for 14-16 hours in this study, the rapid course of development of the embryo dictates that variation in MeHg penetration could result in starkly different timing of toxic exposure. Future studies will employ a recently developed protocol in our lab of permeabilizing the embryo eggshell to circumvent this technical difficulty (Rand 2014; Rand et al 2014). With the above consideration, it is possible that both the motor neuron and the muscle cell are susceptible to the MeHg toxicity, but each has a developmental window of susceptibility that is to some extent non-overlapping.…”

Section: Discussionmentioning

confidence: 99%

The Notch target E(spl)mδ is a muscle-specific gene involved in methylmercury toxicity in motor neuron development

Engel

Rand

2014

Neurotoxicology and Teratology

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Methylmercury (MeHg) is a ubiquitous environmental toxin that has a selective and potent impact on the nervous system, particularly during neural development yet, the mechanisms for its apparent neurodevelopmental specificity are unknown. The Notch receptor pathway has been implicated as a MeHg target in several studies. Notch signaling mediates cell-cell signals in a number of developmental contexts including neurogenesis and myogenesis, where it fundamentally acts to repress differentiation. Previous work in our lab has shown that MeHg causes preferential upregulation of a canonical Notch response gene, E(spl)mδ, in Drosophila embryos. In parallel, MeHg is seen to disrupt outgrowth of embryonic intersegmental motor nerves (ISN), which can be mimicked by expression of activated Notch in embryonic neurons. However, overexpression of E(spl)mδ in developing neurons fails to elicit motor neuron outgrowth defects, pointing to a non-autonomous role for E(spl)mδ in motor axon development. In this study we investigate a role for E(spl)mδ in conveying the toxicity of MeHg in the embryo. We find that endogenous expression of the E(spl)mδ gene localizes to developing somatic muscles in embryos. Notably, E(spl)mδ expression is seen in several muscles that are known synaptic targets for both the ISN and the segmental motor nerve (SN). We also demonstrate that the SN, similar to the ISN, exhibits disrupted axon outgrowth in response to MeHg. E(spl)mδ can induce a SN motor neuron phenotype, similar to MeHg treatment; but, only when E(spl)mδ expression is targeted to developing muscles. E(spl)mδ overexpression in developing muscles also results in aberrant muscle morphology, which is not apparent with expression of the closely related E(spl)mγ in developing muscles. Our data point to a role for the Notch target E(spl)mδ in mediating MeHg toxicity in embryonic development by disrupting the coordinated targeting of motor neurons to their muscle targets.

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“…To allow effective permeabilization of 16-18hr late stage embryos for 1,6-hexanediol treatment, 0-2hr embryos were incubated at 18°C for 32hr, which equals to 16hr development at 25°C (95).…”

Section: Embryo Permealization and 16-hexanediol Treatmentmentioning

confidence: 99%

“…Embryos were dechorionated in 50% bleach for 90s, washed with water for 1 min, and treated with EPS, a d-limonene based solvent with low toxicity (95,96), for 2 min.…”

Section: Embryo Permealization and 16-hexanediol Treatmentmentioning

confidence: 99%

Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin

Lee

Ogiyama

Martins

et al. 2019

Preprint

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1Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in 2 chromosome dynamics and genome stability. However, our current understanding of 3D 3 genome organization does not include PCH domains because of technical challenges 4 associated with repetitive sequences enriched in PCH genomic regions. We investigated the 5 3D architecture of Drosophila melanogaster PCH domains and their spatial associations 6 with euchromatic genome by developing a novel analysis method that incorporates 7 genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, 8 we reveal a hierarchical organization of the PCH domains into distinct "territories." 9 Strikingly, H3K9me2/3-enriched regions embedded in the euchromatic genome show 10 prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2/3 11 enrichment, are likely mediated by liquid-liquid phase separation, and may influence 12 organismal fitness. Our findings have important implications for how PCH architecture 13 influences the function and evolution of both repetitive heterochromatin and the gene-rich 14 euchromatin. 15 16 17Author summary 18 The three dimensional (3D) organization of genomes in cell nuclei can influence a wide 19 variety of genome functions. However, most of our understanding of this critical 20 architecture has been limited to the gene-rich euchromatin, and largely ignores the gene-21 poor and repeat-rich pericentromeric heterochromatin, or PCH. PCH comprises large part 22 of most eukaryotic genomes, forms 3D PCH domains in nuclei, and plays vital role in 23 3 chromosome dynamics and genome stability. In this study, we developed a new method 1 that overcomes the technical challenges imposed by the highly repetitive PCH DNA, and 2 generated a comprehensive picture of its 3D organization. Combined with image analyses, 3 we revealed a hierarchical organization of the PCH domains. Surprisingly, we showed that 4 distant euchromatic regions enriched for repressive epigenetic marks also dynamically 5 interact with the main PCH domains. These 3D interactions are mediated by liquid-liquid 6 phase separation mechanisms, similar to how oil and vinegar separate in salad dressing, 7 and can influence the fitness of individuals. Our discoveries have strong implications for 8 how seemingly "junk" DNA could impact functions in the gene-rich euchromatin. 9 13 comprehensive picture of the 3D structure of PCH domains in late-stage D. melanogaster 14 embryos. Our analysis reveals highly heterogeneous contact frequencies among PCH 15 regions, suggesting hierarchical ordering within the domain. Surprisingly, despite being far 16 from PCH on linear chromosomes, euchromatic loci enriched with H3K9me2/3 can 17 dynamically interact with the main PCH domain, and such interactions show properties 18 consistent with liquid-liquid phase separation and influence individual fitness. Our study 19 demonstrates that the spatial interactions among H3K9me2/3 enriched regions both in 20 PCH and the euchromatic genome...

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A Method of Permeabilization of <em>Drosophila</em> Embryos for Assays of Small Molecule Activity

Cited by 11 publications

References 14 publications

As the fat flies: The dynamic lipid droplets of Drosophila embryos

As the fat flies: The dynamic lipid droplets of Drosophila embryos

The Notch target E(spl)mδ is a muscle-specific gene involved in methylmercury toxicity in motor neuron development

Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin

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